Project Summary The global impact of rickettsial diseases is highlighted by historical records, reemergence of fatal arthropod-borne spotted and typhus fever rickettsioses, and emergence of new pathogens. The intracellular lifestyle of Rickettsia spp. poses immense challenges to research; nevertheless, a transdisciplinary approach alleviates the near intractability of these bacteria to genetic manipulation. This application employs such an approach, targeting one of the most understudied aspects of rickettsial virulence, host-dependent metabolic parasitism. Our recent reconstruction of the Rickettsia metabolic and transport network identified 51 host-acquired metabolites needed to compensate for degraded biosynthesis pathways. Without glycolysis and the pentose phosphate pathway, peptidoglycan (PGN) and lipopolysaccharide (LPS) must be synthesized using host sugars. N-acetylglucosamine 1-P (NAG-1-P), a precursor of both PGN and lipid A disaccharide backbones (as well as other LPS sugars), is usually synthesized by bacteria using the bifunctional enzyme GlmU: the C-terminal acetyltransferase domain generates NAG-1-P from glucosamine-1-P (GlcN-1-P), while the N-terminal uridyltransferase domain converts NAG-1-P to UDP-NAG. Curiously, Rickettsia spp. contain a ?halfling? GlmU enzyme with only the uridyltransferase domain, indicating rickettsiae do not synthesize GlcN-1-P. Given the eukaryotic pathway (synthesizing sialic acid or chitin) generates NAG-1-P from NAG-6-P (not GlcN-1-P), we speculate that rickettsiae import host NAG-1-P and convert it to UDP- NAG using a streamlined enzyme (GlmU-N) tailored to eukaryotic metabolism. Specifically, we hypothesize that rickettsiae uniquely utilize host-derived NAG-1-P for biosynthesis of cell envelope glycoconjugates, and that such metabolite thievery from the host amino sugar biosynthesis pathway is essential for rickettsial intracellular replication and survival. To test our hypothesis, we will characterize rickettsial uptake and metabolism of host NAG- 1-P using sophisticated biochemical techniques (AIM 1), and determine the essentiality of host NAG-1-P pilfering using genetic tools for silencing host and pathogen metabolic genes (AIM 2). Under AIM 1, we will augment host cell amino sugar metabolism (using chemical engineering and isotope supplementation) to monitor synthetic NAG-1-P incorporation into the rickettsial cell envelope. Specifically, we?ll measure azido sugars incorporated into PGN and LPS (via click chemistry and IFA), and 15N-labeled NAG-1-P-derived metabolites incorporated into PGN (via mass spectrometry). Under AIM 2, we will show that rickettsiae must acquire host NAG-1-P for synthesis of glycoconjugates (and thus replication) by using siRNAs to assess the impact of silencing host amino sugar synthesis genes on R. typhi infection, as well as peptide nucleic acid-mediated knockdown of protein expression to determine the functional significance of GlmU-N during early host infection. Collectively, our work will illuminate 1) a novel rickettsial trait (GlmU-N and a unique NAG-1-P transport system) that stands as a promising target for therapeutics aimed at combatting fatal rickettsioses, 2) the impact (pathogenesis) of NAG-1-P pilfering on host cell metabolism, and 3) the importance of NAG-1-P as a critical ingredient for developing a rickettsial axenic media.